The invention pertains to the field of isolation and purification of polynucleotides. In particular, the invention relates to a process for purification of polynucleotides.
The separation and quantification of polynucleotides such as DNA is of critical importance in molecular biology and improved methods are a focus of current interest. One separation method includes size-exclusion chromatography (E. Heftmann, in J. Chromatog. Lib., Vol. 51A, p. A299 (1992)). The disadvantages of this method include low resolution and low capacity. Another separation method is anion exchange chromatography of DNA with tetramethylammonium chloride containing mobile phases as described in European patent application 0 507 591 A2 to Bloch. However, the separation is not strictly size-based, and the resolution is not always adequate. A further disadvantage of methods which rely on binding of anionic DNA includes the required use of high concentrations of nonvolatile salts in the mobile phase; this interferes with subsequent isolation and measurement (e.g. mass spectral analysis) on the separated fragments.
Thus there is a need in the art for a size-based separation process for DNA which has high capacity and resolution, and which does not require use of nonvolatile salts.
Briefly, the instant invention comprises a process for non-specifically binding all of the fragments in a polynucleotide mixture onto a solid binding medium having a hydrophobic surface in the presence of a counterion agent, and the selective release of the fragments based on their size, from smallest to largest, as the concentration of the organic component of the mobile phase is increased. In one embodiment, the binding medium is comprised of beads having a hydrophobic surface. The process does not require use of a high pressure liquid chromatography (HPLC) system and is amenable to scale up or miniaturization.
Accordingly, one aspect of the present invention provides a process for separating a mixture of polynucleotides which is based on the base-pair length of the fragments.
As another aspect of the present invention, there is provided a separation process for a mixture of polynucleotides which can be carried out using binding medium incorporated into a variety of separation configurations including a container such as a column or well.
In another aspect, the present invention provides a separation process for a mixture of polynucleotides which utilizes a hydrophobic binding medium enmeshed in an inert fiber matrix.
In yet another aspect, the present invention provides a simple, inexpensive, and rapid process for separating a mixture of polynucleotide fragments.
In a further aspect, the present invention provides a separation process for polynucleotide fragments which does not require the use of nonvolatile salts.
One embodiment of the instant invention is directed to a batch process for obtaining polynucleotide fragments (such as dsDNA polynucleotides) having a selected size from a mixture of polynucleotide fragments, comprising the steps of
(a) applying a solution of the mixture of polynucleotide fragments and a counterion agent to a binding medium having a hydrophobic surface;
(b) contacting the binding medium with a first stripping solvent and counterion agent, the first stripping solvent having a concentration of organic component, such as acetonitrile, sufficient to release from the binding medium all polynucleotide fragments having a size smaller than the selected size, and removing the first stripping solvent from the binding medium; and
(c) contacting the binding medium with a second stripping solvent having a concentration of organic component sufficient to release from the binding medium the polynucleotide fragments having the selected size, and removing the second stripping solvent from the binding medium.
The binding medium can be rinsed with fresh first stripping solvent following step (b) to remove residual fragments having a size smaller than the selected size therefrom. The binding medium can also be rinsed with fresh second stripping solvent following step (c) to remove residual polynucleotide fragments of the selected size therefrom. The counterion agent preferably is triethylammonium acetate or triethylammonium hexafluoroisopropyl alcohol. The binding medium is preferably porous or nonporous beads having a diameter of from about 1.0 to 1,000 xcexcm. The beads can consist of organic polymer such as a copolymer of vinyl aromatic monomers selected from the group consisting of styrene, alkyl substituted styrene, alpha-methylstyrene and alpha substituted alpha-methylstyrene. Alternatively, the beads can comprise inorganic particles such as silica, silica carbide, silica nitrite, titanium oxide, aluminum oxide, zirconium oxide modified to have a hydrophobic surface. The hydrophobic surface can be an organic polymer supported on the inorganic particle. The hydrophobic surface can be long chain hydrocarbons having from 8-24 carbons bonded to the inorganic particle. Preferably, any residual polar groups of the inorganic particle have been end-capped with trimethylsilyl chloride or hexamethyldisilazane.
The process of the invention is particulary useful in separating the products of a PCR amplification.
The binding medium can be contained within a column, a web or a container. In one embodiment, the medium (such as beads) is contained within a web consisting of an inert fiber matrix and beads enmeshed in the matrix. In a preferred embodiment, the binding medium consists of hydrophobic beads which are contained a polymeric (such as polytetrafluoroethylene) fibril matrix with the ratio of beads to fibril matrix being in the range of 29:1 to 4:1 by weight.
Other aspects and advantages of the present invention are described further in the following detailed description.